Biochemistry 1989, 28, 7356-7363
1356
Synergistic Release of Arachidonic Acid from Platelets by Activators of Protein Kinase C and Ca2+Ionophores. Evidence for the Role of Protein Phosphorylation in the Activation of Phospholipase A2 and Independence from the Na+/HS Exchanger? Stephen P. Halenda,’ Harjit Singh Banga, George B. Zavoico,5 Lit-Fui Lau, and Maurice B. Feinstein* Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06032 Received March 9, 1989; Revised Manuscript Received May 26, 1989
The protein kinase C activators phorbol myristate acetate (PMA), mezerein, oleoylacetylglycerol, and (-)-indolactam V, although without direct effect on arachidonic acid release, greatly enhance the release of platelet arachidonic acid caused by the Ca2+ionophores A23 187 and ionomycin. In contrast, 4a-phorbol 12,13-didecanoate and (+)-indolactam V, which lack the ability to activate kinase C, do not potentiate arachidonate release. Release of arachidonic acid occurs without activation of phospholipase C and is therefore mediated by phospholipase A2. Synergism between P M A and A23187 is not affected by inactivation of the Na+/H+ exchanger with dimethylamiloride. The time course and dose-response for the effect of PMA at 23 OC closely correlate with the phosphorylation of a set of relatively “slowly” phosphorylated proteins (P20, P35, P41, P60), but not the rapidly phosphorylated P47 protein. P20 is myosin light chain, and P41 is probably Gia, but the other proteins have not been positively identified. Depletion of metabolic A T P stores by antimycin A plus 2-deoxyglucose abolishes both protein phosphorylation and the potentiation of arachidonate release by PMA, but does not prevent fatty acid release by the ionophores. Similarly, the kinase C inhibitors H-7 and staurosporine produce, respectively, partial and complete inhibition of PMApotentiated arachidonic acid release and protein phosphorylation, without affecting the direct response to ionophores. These results indicate that protein phosphorylation, mediated by kinase C, promotes the phospholipase A2 dependent release of arachidonic acid in platelets when intracellular Ca2+ is elevated by Ca2+ ionophores. ABSTRACT:
R a t e l e t agonists, such as thrombin and collagen, evoke the rapid release of a substantial fraction of the membrane arachidonic acid that is esterified in phospholipids. Two pathways for the release of arachidonic acid have been described. Arachidonic acid is mobilized in part from phosphoinositides by phospholipase C to yield 1-stearoyl-2-arachidonyldiacylglycerol followed by the concerted action of di- and monoacylglycerol lipases to liberate arachidonic acid (Prescott & Majerus, 1983). A much larger amount of the arachidonic acid release by thrombin is catalyzed by a phospholipase A2 and comes from phosphatidylcholine (Smith et al., 1985). Platelet phospholipase A2 is a Ca2+-dependent enzyme (Baron & Limbird, 1988), and Ca2+ ionophores are effective releasers of arachidonate. Thrombin, in addition to mobilizing Ca2+, can also decrease the Ca2+requirement for arachidonic acid release (Halenda & Rehm, 1987), indicating that there are additional agonist-generated signals that affect phospholipase A2. Some factors proposed to modulate phospholipid hydrolysis by phospholipase A2 include the Na+/H+ exchanger that regulates pHil (Sweatt et al., 1986), GTP-binding proteins (Jelsema & Axelrod, 1987), and lipocortins (calpactins) (Touqui et al., 1986). Activators of protein kinase C, such as tumor-promoting phorbol diesters and diacylglycerol, greatly enhance the release of arachidonic acid by Ca2+ ionophores in platelets (Halenda et al., 1985; Mobley & Tai, 1985). A similar synergism tThis work was supported by National Institutes of Health Grant HL 18937. * Address correspondence to this author. t Present address: Department of Pharmacology, University of Missouri, Columbia, MO. Present address: Bristol Myers Co., Wallingford, CT.
0006-2960/89/0428-7356$01.50/0
between phorbol esters and Ca2+ ionophores also occurs in neutrophils (Volpi et al., 1985; McColl et al., 1986), permeabilized adrenal medullary chromaffin cells (Frye & Holz, 1985), HL-60 cells (Billah & Siegel, 1987), and Madin-Darby canine kidney cells (Parker et al., 1987; Slivka & Insel, 1988; Portilla et al., 1988). However, in some studies there is evidence that kinase C is not involved in the release of arachidonic acid. In these cases the effects of phorbol diesters and diacylglycerol were attributed instead to some type of membrane perturbation affecting the enzyme or its substrates (Hofmann & Majerus, 1982; Dawson et al., 1983; Matsumoto et al., 1988; Tao et al., 1989; Bauldry et al., 1988; Pollock et al., 1986). In this paper we report new evidence indicating that the potentiation of arachidonic acid mobilization by phorbol diesters, and several other kinase C activators, in human platelets is dependent upon protein phosphorylation and is completely eliminated by staurosporine, a potent inhibitor of protein kinase C. EXPERIMENTAL PROCEDURES Materials. Human thrombin ( I 1000 NIH units/mg) from United States Biochemical Corp. was stored in 50% (v/v) glycerol at -20 O C . H-7 and staurosporine were from Seikagaku America, Inc., and Calbiochem, respectively. All other Abbreviations: [Ca2+Ii,cytoplasmic free calcium ion concentration; pHi, intracellular pH; TXB,, thromboxane B,; TXA,, thromboxane A,; HHT, 12-hydroxy-5,8,10-heptadecatrienoicacid; HETE, 12-hydroxy5,8,10,14-eicosatetraenoicacid; PMA, 4P-phorbol 12-myristate 13acetate; PDBu, 4P-phorbol 12,13-dibutyrate; 4a-PDD, 4cu-phorbol 12,13-didecanoate; OAG, I-oleoyl-2-acetyl-glycerol;EGTA, ethylene glycol bis(&aminoethyl ether)-N,N,N‘,N’-tetraaceticacid; HEPES, 4(2-hydroxyethy1)- 1 -piperazineethanesulfonic acid; PIPES, 1.4piperazinediethanesulfonicacid.
0 1989 American Chemical Society
Arachidonic Acid Release from Platelets chemicals were of reagent grade and obtained from various sources. [14C]Arachidonicacid (58.4 mCi/mmol) from Amersham Corp. was stored as a toluene solution under nitrogen at -20 OC. Purity was greater than 98% (thin-layer chromatography, solvent system 1). Ionophores A23 187 and ionomycin, phorbol myristate acetate, phorbol dibutyrate, 4a-phorbol 12,13-didecanoate (4a-PDD), and mezerein were obtained from Sigma Chemical Co., and the (-)- and (+)indolactam V stereoisomers were from LC Services, Inc. These reagents were all stored as single-use aliquots in anhydrous dimethyl sulfoxide at -80 OC. The maximum final concentration of dimethyl sulfoxide present in cell incubations did not exceed 0.3% (v/v) and had no effect of its own on arachidonic acid metabolism. Incorporation of [ l 4 C ] Arachidonic Acid into Platelet Phospholipids. Human platelet concentrates from the American Red Cross, Farmington, CT, were obtained within 24 h of collection. Contaminating blood cells were removed by centrifugation at 125g for 10 min after addition of 1 mM EGTA, and platelets were harvested by centrifugation at 750g for 15 min. Platelets (approximately 2.5 X 1Olo cells) were resuspended in 10 mL of plasma containing 5 WM(2.92 pCi) [ l-14C]arachidonicacid that had been dissolved in 0.025 mL of 100 mM Na2C03,pH 10, before dilution in plasma. The cell suspension was incubated at 37 "C for 60 min and then cooled to rmm temperature. Unincorporated [ 14C]arachidonic acid, amounting to 10-20% of the total radioactivity, was removed by diluting the platelet suspension to 40 mL with nonradioactive plasma (from the same donor) and centrifuging at 7SOg for 15 min. Radiolabeled platelets were resuspended in washing medium containing 145 mM NaCl, 5 mM KC1, 5.5 mM dextrose, 0.2 mM EGTA, and 10 mM PIPES, pH 6.5. After sedimentation at 750g for 10 min, platelets were resuspended to a cell density of 109/mL in incubation medium of 145 mM NaC1, 5 mM KCI, 5.5 mM dextrose, 0.04 mM CaCI,, 1 mM MgCl,, 10 mM HEPES, pH 7.4, and 0.2 mg/mL fatty acid free bovine serum albumin. Intracellular pH was measured with the fluorescent pH indicator BCECF, as described by Zavoico et al. (1986). Analysis of [ ''C]Arachidonic Acid and Metabolites. Agents that affect arachidonic acid mobilization were added to aliquots (0.5 mL) of platelet suspension incubated in siliconized glass tubes at 23 OC. Incubations were terminated by adding 1.88 mL of ice-cold methanol/chloroform (2:l v/v). Phases were separated by adding 0.6 mL each of chloroform and 0.1% (v/v) formic acid followed by vigorous mixing and centrifugation to extract arachidonic acid and metabolites. The lower chloroform-rich phases were collected and concentrated under nitrogen, and the radioactive arachidonic acid and metabolites were separated by thin-layer chromatography using solvent system 1 [chloroform/methanol/acetic acid/water (90:8:1:0.8 v/v)], in which phospholipids remain at the origin. Analysis of [ 14C]Arachidonate-and [32P]Phosphate-Labeled Phospholipids. Phospholipids were analyzed as previously described (Halenda et al., 1985) after extraction of platelets (0.5 mL) with 1.88 mL of ice-cold methanol/chloroform/HCl (100:50:1 v/v). Phases were separated with 0.6 mL each of chloroform and 2 mM KCI. After concentration of the chloroform-rich phases under nitrogen, the phospholipids were chromatographed on Merck silica gel 60 thin-layer plates with solvent system 2 [chloroform/methanol/20% (w/v) aqueous methylamine (60:36: 10 v/v)]. Radioactive lipid bands located by autoradiography and identified by comigration with standards were scraped and quantitated by scintillation counting. All extraction solvents contained 0.01 mg/mL of
Biochemistry, Vol. 28, No. 18, 1989
7357
the antioxidant butylated hydroxytoluene. [ 14C]Arachidonic acid release is expressed either as counts per minute (mean percent SD for triplicate determinations, unless otherwise indicated) from a single representative experiment or, when averaged results from more than one experiment are shown, as percent release of [I4C]arachidonic acid: percent release (cpm) = 100 X cpm (arachidonic acid + TXB, HHT + HETE)/cpm (phospholipids arachidonic acid TXB2 H H T + HETE). Measurement of the total amount of [ 14C]arachidonic acid released from phospholipids allows comparison of data from platelets from different donors, which incorporated [ 14C]arachidonic acid to varying extents. [32P]Phosphate-labeled phosphoinositides and phosphatidic acid were analyzed as previously described (Zavoico et al., 1985). Protein Phosphorylation. Suspensions of washed platelets (3 X lo9 to 10l0/mL) were incubated for 45 min at 37 "C with 0.2 mCi/mL of ~rtho[~~P]phosphoric acid (carrier-free, New England Nuclear, Boston, MA). Buffer containing 0.2 mM EGTA was added, and the suspension was centrifuged for 10 min at 750g. The platelet pellet was resuspended in incubation buffer containing 1 mM MgC12 and 40 pM CaC12 to a concentration of 5 X lo8 cells/mL and then diluted to 2.5 X los cells/mL in the experiments. Reactions were stopped with a solution of 2.5% dithiothreitol, 2% sodium dodecyl sulfate, 12.5% glycerol, and a trace of bromophenol blue and then placed in a boiling-water bath for 5 min. The samples were electrophoresed essentially as described previously (Feinstein et al., 1983) on straight 10%or 5-1596 gradient polyacrylamide gels containing 0.1% SDS. The gels were stained with Coomassie Brilliant Blue R, destained, and dried, the phosphorylated proteins located by radioautography, and the radioactive gel bands cut out, digested, and counted by liquid scintillation spectrometry.
+
+ +
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RESULTS Potentiation of Ionophore-Induced Arachidonic Acid Release by Activators of Protein Kinase C. Ca2+ ionophores release arachidonic acid in platelets treated with aspirin plus ADP scavengers, which prevents activation of phospholipase C via thromboxane and ADP receptors (Rittenhouse & Horne, 1984). Platelets treated in this way did not hydrolyze phosphatidylinositol4,5-bisphosphateor produce phosphatidic acid from diglyceride in response to A23187, in sharp contrast to the receptor-agonist thrombin (Figure IA). These data, and those previously presented (Halenda et al., 1985), show that the release of arachidonic acid by ionophores plus PMA is most probably catalyzed solely by phospholipase A,. A23187 stimulated the appearance of free [14C]arachidonicacid and its radioactive metabolites, TXB,, HHT, and I2-HETE, in washed platelet suspensions. Figure 1 shows the total amount of [I4C]arachidonic acid (arachidonic acid plus metabolites) released from phospholipids as a function of ionophore concentration. Release of radioactive fatty acid was just detectable at 0.5 pM A23187 (0.5 nmol of A23187/109 platelets), and the maximal release (approximately 20%) was attained at about 5 pM ionophore (5 nmol of A23187/109 platelets). In untreated platelets the free [14C]arachidonic acid plus metabolites was only 0.19% (*0.01% SE; eight exp) of the total radioactive arachidonic acid in phospholipids. PMA had two effects on arachidonic acid release (Figure 1B). One effect was to increase sensitivity to the ionophore; e.g., at a minimally effective concentration of ionophore (Le., 0.5 pM) pretreatment with 100 ng/mL PMA increased total arachidonic acid release by about 8-f0ld, and maximum release occurred at 1 pM rather than 5 pM A23187. Second, PMA
7358
Biochemistry, Vol, 28, No. 18, 1989
Halenda et al.
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Effects of PMA on A23187-induced release of arachidonic acid and formation of its metabolites. Washed platelets as in Figure 1 (but not treated with aspirin) were stimulated with indicated concentrations of A23187 alone ( 0 )or after retreatment with 160 nM PMA (0). [14C]Arachidonicacid and * C-labeled metabolites were assayed as described under Experimental Procedures. Note the differences in scale of radioactivity for different metabolites. FIGURE 2:
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(A) Phosphatidylinositol 4,5-bisphosphateand phosphatidic acid in platelets stimulated with A23187 or thrombin. Washed platelets ( 109/mL)labeled with [3ZP]phosphatewere treated with 1 mM aspirin for 20 min, incubated in the presence of 5 mM creatine phosphate/creatine phwphokinase (40units/mL), and then stimulated or 1 unit/mL thrombin (0,m) for the times with 1 pM A23187 (0,O) indicated. [32P]Phosphatidylinositol4,5-bisphosphate (circles) and [32P]phosphatidicacid (squares) were analyzed as described under Experimental Procedures. (B) Potentiation of arachidonic acid release by PMA. Washed platelets ( 109/mL), prelabeled with [I4C]arachidonic acid, were stimulated for 5 min with indicated concentrations of A23 187. Total [14C]arachidonicacid released (free arachidonate plus all radioactive metabolites) was measured as described under Experimental Procedures: ( 0 )A23187; (0)treated with PMA 100 ng/mL (160 nM) for 10 min prior to A23187. Temperature was
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increased the maximum amount of arachidonic acid that could be released from phospholipids by 2-fold (from 20% to 40%) (Figure 1B). Similar results were obtained with the Ca2+ ionophore ionomycin (see below). PMA also enhanced the formation of arachidonic acid metabolites (Figure 2). We previously showed that PMA did not affect the metabolism of exogenous arachidonic acid or its esterification into phospholipids (Halenda et al., 1985), so the production of the metabolites solely reflects the much greater availability of substrate (free arachidonic acid). At 0.5 pM A23 187 PMA increased free arachidonic acid 5-fold and thromboxane B2 and HHT by more than 10-fold. At low concentrations of A23181 the products of the cyclooxygenase/thromboxane synthetase pathway (thromboxane B2 plus HHT) roughly equaled the amount of 12-HETE formed by the lipoxygenase pathway. At higher concentrations of A23187 free arachidonic acid plus 12-HETE comprised the greatest portion (Le., 75%) of the total amount of arachidonic acid released after 5 min. In some cultured cells PMA increases the spontaneous appearance of arachidonic acid metabolites in the medium, which is a very slow process compared to the explosive events in platelets. In these cells, PMA stimulates synthesis of prostaglandin G / H synthase (Wu et al., 1988; Goerig et al., 1988) and thromboxane synthase (Goerig et al., 1988), and this effect is blocked by cycloheximide. As expected, in anucleate platelets, cycloheximide (10
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Effect of indolactam V stereoisomers on arachidonic acid release. [ 14C]Arachidonicacid labeled platelets were treated as follows: (solid bars) not treated with indolactams, (C) controls, (A23) 1 KM A23187 for 5 min; (open bars) (ILV) 20 pM (-)-indolactam V for 10 min, (A23 + ILV) pretreated with (-)-ILV (20 pM) for 10 min prior to A23187, (A23,ILV + Stp) pretreated with 1 pM staurosporine for 3 min prior to indolactam and then A23187; (hatched bars) same experiment as open bars but using (+)-indolactam V. Total [I4C]arachidonic acid (plus metabolites) was measured as in Figure 1B. pg/mL) did not alter the enhancement of arachidonic acid release by PMA (not shown). The potentiating effect of PMA was mimicked by the synthetic diacylglycerol OAG and phorbol dibutyrate, but not by the phorbol diesters 4a-PDD or 4a-PMA, which do not stimulate protein kinase C (Halenda et al., 1985; Halenda & Rehm, 1987). These results implicated protein kinase C in the release of arachidonic acid. To further test this hypothesis, we employed other kinase C activators, Le., mezerein, a non-phorbol ester diterpene lacking a diacylglycerol-like moiety (Miyake et al., 1984), and (-)-indolactam V, which is similar to the active structural element of tumor-promoting teleocidins (Fujiki et al., 1984). Mezerein potentiated arachidonic acid release, and activated protein phosphorylation, to about the same extent as PMA (see Table I and Figure SC). Figure 3 shows experiments using indolactams. (-)-Indolactam V (20 pM), an activator of kinase C, produced a 4.3-fold increase of arachidonic acid release by 1 &I A23187, similar to the potentiation by PMA at the same concentration of ionophore (Figure I B). In contrast, the optical stereoisomer (+)-indolactam V, which does not stimulate kinase C, also did not potentiate arachidonic acid release (Figure 3).
Biochemistry, Vol. 28, No. 18, 1989 7359
Arachidonic Acid Release from Platelets
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Dimethylami I or ide [uM]
FIGURE 4: Potentiation of arachidonic acid release by PMA in latelets treated with dimethylamiloride. Platelets prelabeled with $Clarachidonic acid were stimulated with 1 pM ionomycin alone).( or ionomycin after 160 nM PMA (0) in the presence of indicated concentrations of dimethylamiloride. Controls (H) received equivalent amounts of vehicle (DMSO). Percent [ 14C]arachidonicacid release is calculated as described under Experimental Procedures. [Insert (above)] Effect of ionomycin alone (I) or ionomycin added after 100 pM dimethylamiloride (D) on intracellular pH measured with the fluorescent probe BCECF. A decrease in fluorescence indicates a fall of pHi. The initial pHi was 7.0.
An Inhibitor of Na+/rr' Exchange Does Not Affect Enhancement of Arachidonic Acid Release by PMA. Stimulation of the Na+/H+ antiporter by PMA might facilitate arachidonic acid release by elevating the pHi to a more favorable level for phospholipase A2 activity (Banga et al., 1986; Baron & Limbird, 1988). When platelets were pretreated with 0.1-1 mM dimethylamiloride, the Na+/H+ antiporter was totally inactivated (Zavoico et al., 1986). Under these conditions dimethylamiloride did not hinder potentiation of arachidonic acid release by PMA, despite a fall of more than 0.2 pHi caused by A23 187 or ionomycin (Figure 4). The potentiation of arachidonic acid release by PMA is therefore not dependent on a rise in pHi or stimulation of the Na+/H+ antiporter. Effects of PMA on Protein Phosphorylation. We used an incubation temperature of 22-23 "C to slow responses, enabling temporal resolution of events that are nearly concurrent at 37 "C. At 37 "C the full potentiation of arachidonic acid release by 100 ng of PMA/ lo9 platelets occurs in only 2-3 min (not shown), whereas at 23 O C half-maximal potentiation develops in about 5 min and the full effect is attained in about 15 min (Halenda et al., 1985). PMA, mezerein (Figure 5 ) , (-)-indolactam V, OAG, and thrombin (not shown) stimulated phosphorylation of the 47-kDa protein (P47), 20-kDa myosin light chains (P20), and unidentified polypeptides of 60-65, 41, (P41), and 35-37 kDa (P35). The rate and dose-response relationship for PMA-induced phosphorylation of P47 at 23 "Cdiffered substantially from potentiation of arachidonic acid release (Halenda et al., 1985). Other proteins were phosphorylated more slowly than P47; e.g., P20, P35, and P41 phosphorylation was low when P47 phosphorylation was maximal (Figure 5A,B). Most interesting, both the time course and dose-response for potentiation of arachidonic acid release by PMA at 23 "C (Halenda et al., 1985) corresponded closely to phosphorylation of P41, P35, and P20 induced by PMA (Figure 5 ) . Protein Kinase C Inhibitors Prevent Potentiation of Arachidonic Acid Release by PMA. The kinase C inhibitor H-7
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1
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5: Stimulation of protein phosphorylation by PMA and
mezerein. (A) Time course of phosphorylation of P47 (O), P41 (0), and P20 (H) by 160 nM (100 ng/mL) PMA. (B) Dose-response for protein phosphorylation induced by PMA (10-min incubation). (C) Phosphorylation of platelet roteins by PMA and mezerein. Washed platelets prelabeled with [3 Plphosphate were treated as follows and then analyzed after SDS-PAGE by radioautography: (lane 1) 160 nM (100 ng/mL) PMA, 10 min; (lane 2) 100 nM mezerein, 10 min; (lane 3) 1 pM mezerein, 10 min; (lane 4) 160 nM PMA, 1 min; (lane 5 ) 100 nM mezerein, 1 min; (lane 6) 1 pM mezerein, 1 min; (lane 7) no PMA or mezerein. Temperature was 23 OC.
4
Table I % inhibition of arachidonic acid releasea
mezerein PMA (100 nM) (100 nM) OAG (40 pg/mL) 50 6.7 f 3.9 (4) 8.4 (2) 30.9 (2) 100 14.3 f 6.5 (6) 36.7 f 8.2 (3) 54.8 f 8.6 (6) 150 23.5 (2) 200 33.0 f 8.6 (6) 71 (1) 55.4 f 8.1 (3) Washed platelets were incubated in the presence of H-7 for 5 min and then exposed to kinase C activator for an additional 5 min before 0.5 pM A23187 was added. After 5 min, reactions were terminated and arachidonic acid plus metabolites assayed. A23187 (0.5 pM, 5 min) released 6.1% (f0.7, n = 14) of platelet radioactive arachidonate. The kinase C activators increased total arachidonic acid release as follows: PMA 276% (f32%, n = 12), mezerein 181% (f18%, n = 4). and OAG 193% (12395, n = 7). The data are expressed as percent inhibition of the potentiated arachidonic acid release (fSE,number of experiments). H-7 did not inhibit A23187 alone. H-7 (pM)
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(50-200 pM) only partially prevented the potentiation of arachidonic acid release by PMA, OAG, and mezerein (Table I). However, staurosporine, a more potent and effective inhibitor of platelet kinase C activity (Watson et al., 1988), completely abolished the potentiation by kinase C activators, including PMA (Figure 6) and (-)-indolactam V (Figure 3), with little or no inhibition of the response to A23 187 alone (Figures 3 and 6). Staurosporine inhibited PMA-induced phosphorylation of P60-65, P47, P41, P35, and P20 to a
Biochemistry, Vol. 28, No. 18, 1989
7360
Halenda et al.
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6: Effects of staurosporine on PMA-potentiated release of [14C]arachidonicacid. (A) (open bars) DMSO for 10 min followed by 1 pM A23187 for 5 min; (hatched bars) 160 nM PMA (in DMSO) for 10 min prior to addition of A23187. Staurosporine (Stp) was added at indicated concentrations for 3 min prior to DMSO PMA. Total arachidonic acid ( lus metabolites) released was measured. (B) Time course of total [ I BClarachidonic acid release by A23187 alone (0) or after pretreatment with 0.1 pM ( 0 )or 1.0 pM (A)staurosporine. FIGURE
*
comparable degree (not shown). The dose-response for inhibition of PMA-induced secretion of dense granule ATP by staurosporine (Figure 7A) was similar to that for inhibition of protein phosphorylation (Figure 7B) and arachidonic acid release (Figure 6A). Metabolic Inhibitors Prevent PMA-Potentiated Arachidonic Acid Release and Protein Phosphorylation. Blockade of oxidative phosphorylation and glycolysis by incubation of platelets with 4 pg/mL antimycin A (or carbonyl cyanide m-fluorophenylhydrazone) plus 32 m M 2-deoxyglucose decreased the metabolic pool of ATP by 75% in 30 min (Holmsen et al., 1982). This treatment of our platelets was without effect on arachidonic acid release caused by A23 187 (or ionomycin) alone (Figure 8), but it nearly abolished protein phosphorylation by PMA and totally blocked PMA's enhancement of arachidonic acid release by 0.5-1.0 pM A23187 (or ionomycin, not shown). Metabolic inhibitors were earlier shown to prevent arachidonic acid release by thrombin (but not A23187) (Rittenhouse-Simmons & Deykin, 1977). The depletion of ATP (and GTP) would be expected to impair the Ca2+-mobilking effect of thrombin (but not A23187) by reducing the amount of available phosphatidylinositol 4,5-bisphosphate and its GTP-dependent hydrolysis necessary for inositol 1,4,5trisphosphate production.
DISCUSSION The tumor promoters PMA and phorbol dibutyrate and the diacylglycerol OAG strongly potentiated arachidonic acid release by Ca2+ ionophores without any direct effect by
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FIGURE 7: Inhibition of PMA-induced ATP secretion and protein phosphorylation by staurosporine. (A) Platelets (109/mL) were stimulated by 160 nM PMA, and ATP secretion was measured with luciferin/luciferase in a lumiaggregometer. Controls, treated with DMSO alone, secreted approximately 20 nmol of ATP/109 platelets. Staurosporine (in DMSO), at concentrations indicated, was added 3 min prior to PMA. Temperature was 22 OC. (B) Phosphorylation of P47; (open bar) 160 nM PMA for 10 min; (hatched bars) pretreatment with staurosporine (Stp) at indicated concentrations (0.1 and 1.0 gM), added 3 min before PMA). (C) Time course of phosphorylation of P47 by 160 nM PMA (0),PMA + 0.1 pM Stp ( O ) , and PMA + 1.0 pM Stp (A). Note that after the initial burst of phosphorylation, dephosphorylation ensues in the presence of staurosporine. P47 was measured because of greater 32Pincorporation. Results were similar for other substrate proteins as well.
themselves (Halenda et al., 1985; Mobley & Tai, 1985; Toqui et al., 1986; Halenda & Rehm, 1987; this study). Since 4aphorbol diesters were ineffective, it appeared that kinase C was involved in the potentiation of arachidonic acid release by ionophores. To test this hypothesis more thoroughly, we used two other kinase C activators, which differ significantly from the phorbol diesters in chemical structure. Mezerein and (-)-indolactam V were also found to facilitate release of arachidonic acid in intact platelets. In contrast, (+)-indolactam V has very low affinity for kinase C (Fujiki et al., 1984) and did not potentiate arachidonic acid release by A23187. The inactivity of the latter compound is especially significant because it is the optical stereoisomer of the strong potentiator (-)-indolactam V. To further understand the mechanism of action of these agents, the effects of PMA were more thoroughly investigated. PMA has four characteristic effects on arachidonic acid release by ionophores: ( 1 ) enhancement of release requires that ionophores elevate [Ca2+Iito some threshold level, which by itself is sufficient to initiate only a small but detectable release of fatty acid; (2) at concentrations of ionophores that produce minimal effects, PMA can increase arachidonic acid release
Biochemistry, Vol. 28, No. 18, I989
Arachidonic Acid Release from Platelets
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LA 23 I8dIJIM
FIGURE 8: Inhibition of protein phosphorylation and PMA-potentiated arachidonic acid release by metabolic inhibitors. Washed platelets prelabeled with [32P]phosphateand [14C]arachidonatewere incubated 30 min at 37 'C with deoxyglume (DOG) plus antimycin A (antiA) to deplete the metabolic pool of ATP. (A) Densitometric scans of radioautograph of 32P-labeledproteins from platelets incubated 10 min with 160 nM PMA under normal conditions (a) or after 30 min of incubation with metabolic inhibitors (b). (B) ['4C]Arachidonic acid release: Normal platelets plus 1 pM A23187 ( O ) , A23187 after 160 nM PMA (0); metabolically inhibited platelets plus 1 pM A23187 (I)and A23187 after PMA ( 0 ) . Same protocol as Figure 1B.
as much as 10-fold, with a corresponding increase in the production of thromboxane; (3) at saturating concentrations of ionophore, PMA can further increase the maximum amount of arachidonic acid release by 2-fold; and (4) PMA does not affect the metabolism of free arachidonic acid. We also previously reported that PMA did not affect the incorporation of arachidonic acid into platelet phospholipids (Halenda et al., 1985). However, Fuse et al. (1989) recently reported that PMA caused inactivation of two enzymes that are responsible for uptake of arachidonate and its esterification into platelet phospholipids (Le., arachidonyl-coenzyme A synthase and arachidonyl-CoA lysophosphatide acyltransferase). Inactivation of these enzymes would prevent the reesterification of released arachidonate and might account for an apparent potentiated release of arachidonate by the combined action of PMA and ionophores. We have reinvestigated this problem and find no interference by PMA with arachidonate uptake into platelets or its incorporation into phospholipids; indeed, arachidonate incorporation into PI and PA was increased.2 Furthermore, 100 pM 8,11,14-eicosatrienoic acid totally blocked uptake of arachidonic acid (5 pM) into platelets, but unlike PMA had no effect on the release of arachidonic acid by A23187. From these data we conclude that interference with the reincorporation of arachidonic acid released by ionophore is not a valid explanation for the potentiation of arachidonate release by PMA. In Ca2+-depleted "ionophore-permeabilized" platelets arachidonic acid is released as a function of Ca2+in the medium over the range 0.1-10 pM, similar to the concentration attained in the cytoplasm of stimulated platelets (Rehm & Halenda, 1987). Under these conditions PMA or thrombin cause a leftward shift (potentiation) of the Ca2+dose-response curve and an increase of the maximum amount of arachidonic acid released. This effect is similar to the leftward shift of the ionophore dose-response curves by PMA by intact platelets and shows that PMA, although lacking the ability to directly
* Halenda, Banga, Lau, and Feinstein, unpublished results.
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stimulate phospholipase A2, potentiates the ability of elevated calcium to cause phospholipid hydrolysis. On the basis of these findings the initial focus of our investigations was on two known mechanisms of action of phorbol diesters, Le., increase of intracellular pH (pHi) mediated by the amiloride-sensitive Na+/H+ exchanger and phosphorylation of proteins mediated by protein kinase C. Intracellular pH can modulate the release of arachidonate by weak platelet agonists (e.g., epinephrine, ADP) and low concentrations of thrombin (Sweatt et al., 1986). Because arachidonic acid release was inhibited by amiloride derivatives or by removal of extracellular Na+, Sweatt et al. concluded that the Na+/H+ exchanger was important for arachidonic acid release by Ca2+-mobilizing agonists. The Na+/H+ exchanger is responsible for a sustained alkalinization of the cytosol after stimulation by thrombin (Zavoico et al., 1986), which would favor hydrolysis of phospholipids because of the alkaline pH optimum of phospholipase A2 (Baron & Limbird, 1988). Amiloride derivatives or lack of extracellular Na+ converts the normal increase of pHi (approximately 0.15 unit) caused by thrombin to a sustained fall of 0.35 pHi below the normal resting level of pHi 7.0 (Zavoico et al., 1986). This fall of pHi could inhibit responses to weak agonists, and similarly stimulation of platelet Na+/H+ exchanger by PMA (Siffert & Scheid, 1986; Siffert & Akkerman, 1988) might account for its potentiation of arachidonic acid release. However, PMA's potentiation of arachidonic acid release by ionophores is not inhibited by the potent Na+/H+ exchange inhibitor dimethylamiloride, despite the substantial fall of pHi caused by ionophore under these conditions. This demonstrates conclusively that PMA/ionophore synergism is not mediated by the Na+/H+ antiporter. Indeed, treatment with PMA appears to make phospholipase A2 behave as if it were at high pH and may ultimately affect enzyme conformation and activity in a similar way. It is important to point out that our results do not dispute the findings of Sweatt et al. (1986) regarding weak agonists, and in some cases inhibition of the Na+/H+ antiporter may also attenuate the mobilization of Ca2+ (Sweatt et al., 1986; Zavoico et al., 1986; Siffert & Akkerman, 1988). Potentiation of arachidonic acid release by PMA could also be mediated by protein phosphorylation, to produce an activator, or to inhibit an antagonist, of phospholipase A> Several studies in other types of cells (e.g., neutrophils) did not support the involvement of kinase C, because the kinase C inhibitor H-7 failed to block the response to PMA (Billah & Siegel, 1987; Matsumoto et al., 1988; Tao et al., 1989). It is also reported that the alkylacyl analogue of OAG, l-O-(9-octadecenyl)-2-acetylglycerol,stimulates arachidonic acid release in neutrophils, although it lacks the ability to activate kinase C (Bauldry et al., 1988). These studies suggest that diacylglycerols and phorbol diesters have nonspecific effects on membrane properties or directly enhance phospholipase activity (Dawson et al., 1983). It should be borne in mind, however, that agonist-induced arachidonic acid mobilization and metabolism in neutrophils differ quite substantially from that in platelets and may therefore be subject to different types of control. In platelets, we believe a strong case can be made for involvement of kinase C (or some other kinase) in the response to PMA because the effect is (a) produced by kinase C activators of several different chemical classes, but not by closely related compounds or stereoisomers that do not stimulate kinase C, (b) correlated temporally, and with respect to dose, with PMA-induced phosphorylation of certain proteins, (c) abolished by depletion of ATP, and (d) partially or com-
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pletely inhibited by H-7 and staurosporine, respectively. The efficacy of these inhibitors against arachidonic acid release is well correlated with their potency to block protein phosphorylation. H-7, which is a weak and only partial inhibitor of protein kinase C dependent protein phosphorylation in platelets (Watson et al., 1988), only partially inhibited the potentiation of arachidonic acid release by OAG, mezerein, and PMA without affecting arachidonic acid release by A23187 alone. In contrast, staurosporine, a much more potent inhibitor of kinase C in platelets (Watson et al., 1988), was able to totally abolish potentiation of arachidonic acid release by PMA and (-)-indolactam V. Over a concentration range of 0.02-1 .O pM, staurosporine produced equivalent inhibition of PMA-induced secretion, protein phosphorylation, and arachidonic acid release. From the selective action of the metabolic inhibitors and staurosporine we deduce that a protein kinase may mediate the action of PMA, but not the direct effect of Ca2+ on phospholipase A2. Although staurosporine is reportedly most selective against the serine/threonine kinase C, we cannot discount the possibility that it may affect other kinases, such as platelet tyrosine kinase (Golden & Brugge, 1989). The situation is further complicated by the fact that platelets have two classes of phorbol diester binding sites (Cech et al., 1987) and at least two kinase C isozymes, one of which is quite insensitive to Ca2+ (Tsukuda et al., 1988). Phosphorylation of the major platelet phosphoprotein P47 does not correlate temporally or in dose-response with PMA-induced arachidonic acid release [see also Halenda et al. (1985)l. However, reducing the temperature to 21-23 OC causes both the time course of potentiation of arachidonic acid release by PMA and the protein phosphorylation to be greatly slowed, permitting greater resolution of these events. A group of proteins, which include P41, P35, P20, and possibly P60, are slowly phosphorylated compared to P47. Their phosphorylation closely corresponded both in time course and in dose-response to the potentiation of arachidonic acid release by PMA. P20 is the myosin light chain that is phosphorylated by kinase C (Nishikawa et al., 1984) and Ca2+/calmodulin-dependent protein kinase. The 35-37-kDa polypeptide is similar in molecular weight and p l (pH 27.5, not shown) to platelet lipocortin, a protein that inhibits phospholipid hydrolysis by pancreatic phospholipase A2 in vitro (Toqui et al., 1986). Lipocortins (calpactins) can be inactivated by phosphorylation by kinase C (Schlaepfer & Haigler, 1988; Varticovski et al., 1988) or tyrosine kinases (Varticovski et al., 1988; Toqui et al., 1986). There are conflicting data about the biochemical mechanism of action of lipocortins (Davidson et al., 1987; Haigler et al., 1987; Huang et al., 1987; Nevalainen & Evilampi, 1984; Parente & Flower, 1985), and their physiological significance as regulators of arachidonic acid release remains to be established. P41 is phosphorylated in intact PMA-treated platelets and on 2-dimensional gels (SDS-PAGE/isoelectric focusing) migrates identically with the ADP-ribosylated pertussis toxin substrate 41 kDa G i a (Halenda et al., 1986). The GTPbinding protein G i a is phosphorylated by kinase C in vitro (Katada et al., 1985), but its role in platelet arachidonic acid release is not known. GTP-binding proteins have been indirectly implicated in platelet arachidonic acid release (Nakashima et al., 1987a; Fuse & Tai, 1987). In permeabilized platelets GTPrS only slightly increased arachidonate release, but it potentiated the much larger release by thrombin (Nakashima et al., 1987b), suggesting an interaction between a
Halenda et al. GTP-binding protein and some other signal(s) generated by thrombin. A more definitive link between G-proteins and arachidonic acid release has been provided by experiments utilizing purified G-protein components. The pr subunits of the GTP-binding protein transducin are reported to stimulate phospholipase A2 activity (Jelsema & Axelrod, 1987; Kim et al., 1989). It has been suggested that 07 may stimulate phospholipase A2 activity by counteracting calpactin (Jelsema, 1989). Various G a subunits may also directly or indirectly affect phospholipase A2 (Jelsema, 1989). Although our evidence supports the hypothesis that kinase C can potentiate arachidonic acid release by Ca2+ionophores in platelets, the enzyme's role in responses mediated by receptor agonists has not been established. In one study the kinase C antagonist H-7 failed to prevent arachidonic acid release by thrombin in intact platelets (Fuse & Tai, 1988), but inhibition was observed by other investigators when the intracellular free Ca2+was fixed (Halenda & Rehm, 1987). In intact platelets we find that the more potent kinase inhibitor staurosporine slightly, but consistently, enhanced arachidonic acid release by t h r ~ m b i n . ~ Staurosporine most likely prevents the negative feedback that is normally exerted on thrombininduced phosphoinositide hydrolysis and CaZ+mobilization (Rittenhouse & Sasson, 1985; Zavoico et al., 1985; Yoshida & Nachmias, 1987). This negative feedback, which results from activation of kinase C caused by the stimulus-induced formation of diacylglycerol, may serve to limit arachidonic acid release. The existence of this kinase C mediated negative feedback pathway complicates the attempt to demonstrate a direct stimulatory role of the enzyme in receptor-mediated arachidonic acid release. Also, we must consider the possibility that thrombin operates to release arachidonic acid through a different pathway than PMA plus Ca2+ ionophores, perhaps involving a protein kinase that is unaffected by staurosporine or GTP-binding protein components (Jelsema, 1989). In conclusion, the magnitude of the synergism between ionophores and kinase C activators demonstrates that although kinase C is not necessary for arachidonic acid release mediated by elevated Ca2+,the enzyme is a potentially important regulator of phospholipase A2, either directly or indirectly. Even if the responses to PMA/Ca2+ ionophore and thrombin proceed by somewhat different pathways, it is possible that they ultimately converge at a common site, or interacting sites of action, such as some inhibitor, or activator (Park, 1989), of phospholipase A2. REFERENCES Banga, H. S., Simons, E. R., Brass, L. F., & Rittenhouse, S. E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9197-9201. Baron, B. M., & Limbird, L. E. (1988) Biochim. Biophys. Acta 971, 103-111. Bauldry, S. A., Wykle, R. L., & Bass, D. A. (1988) J . Biol. Chem. 263, 16787-16795. Billah, M. M., & Siegel, M. I. (1987) Biochem. Biophys. Res. Commun. 144, 683-691. Cech, P., Cohen, L. E., Szklarek, D., & Lehrer, R. I. (1987) Thromb. Res. 45, 827-837. Davidson, F. F., Dennis, E. A,, Powell, M., & Glenney, J. R., Jr. (1987) J . Biol. Chem. 262, 1698-1705. Dawson, R. M. C., Hemington, N. L., & Irvine, R. F. (1983) Biochem. Biophys. Res. Commun. I 17, 196-201. Feinstein, M. B., Egan, J. J., & Opas, E. E. (1983) J . Biol. Chem. 258, 1260-1267. Lau, Banga, and Feinstein, unpublished results.
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